CN102778569B - Method for detecting insecticidal crystal protein Cry1Ab based on micro-cantilever - Google Patents
Method for detecting insecticidal crystal protein Cry1Ab based on micro-cantilever Download PDFInfo
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Abstract
The invention discloses a method for detecting an insecticidal crystal protein Cry1Ab based on a micro-cantilever. The method comprises the following steps of modifying 11-mercaptoundecanoic acid, 6-mercaptohexanoic acid or 3-mercaptopropionic acid serving as a sulfhydrylization reagent onto a gold film of the micro-cantilever; and activating carboxyl of the sulfhydrylization reagent with 1-ethyl-(3-dimethyllaminopropyl)carbodiie hydrochlide (EDC.HCl) and N-hydroxysuccinimide (NHS), so that the activated carboxyl is combined with amino of an insecticidal crystal protein Cry1Ab antibody to obtain a micro-cantilever on which the insecticidal crystal protein Cry1Ab antibody is modified. According to the method, a good effect is achieved when the micro-cantilever on which the insecticidal crystal protein Cry1Ab antibody is modified is applied to detection of the insecticidal crystal protein Cry1Ab. The method does not require marking, is easy to operate, has high sensitivity, is possibly used for replacing the conventional enzyme-linked immunosorbent assay method, and plays a very important role in analyzing and detecting genetically modified species.
Description
Technical field
The present invention relates to a kind of method detecting insecticidal crystal protein Cry1Ab based on micro-cantilever, be specifically related to a kind of method and application of micro-cantilever finishing insecticidal crystal protein Cry1Ab antibody.
Background technology
Cry1Ab albumen is the toxalbumin that one comes from the gene expression of Dipel [Bacillus Thuringiensis (Bt)].Bacillus thuringiensis is gram-positive bacteria, and to be a class to strong, the lethal speed of insect virulence fast and to the avirulent entomopathogen of pest natural enemy.Thalline can form insecticidal crystal proteins in the stable growth phase, also known as insecticidal crystal protein (ICP) or D2 endotoxin.ICP, by Cry gene and Cyt gene code, has stronger toxicity to sensitive insect, and to higher mammal and people's nontoxicity.CrylAb belongs to Cry gene family, is the toxalbumin (Microbiol.Rev.1989,53,242) of Bt gene expression.Bt has become the strong substitute of chemical synthetic pesticide in the control of agricultural pests, injurious forest-insect and sanitary insect pest, the gene source that it or transgenic pest-resistant engineered plant are important.Bt albumen is measured quickly and accurately, provides technical support by for the Pest management of breeding, genetically modified plants and genetic marker.Bt method of protein detection common at present has the PCR method (Anal.Bioanal.Chem.2003 detected based on nucleic acid DNA, 373, 985) with based on enzyme-linked immunosorbent assay (the ELISA) (Nature of Protein Detection, 1999, 402, 480), PCR method can carry out Accurate Measurement to transgene component from crop seed in the whole process of crop maturity, not by the impact of the disposal route of sample, but easily there is the problem such as false negative and false positive, for the transgenic product through highly processing and derivant thereof, as vegetable oil, sugar or starch etc., they are not containing any DNA composition, therefore PCR method is difficult to realize the detection to this type of enetically modified food, MBP enzyme linked immuno-adsorbent assay is a kind of Fast Detection Technique based on antigen and the specific binding of antibody and the efficient catalytic characteristic of zymolyte, has the features such as quick, sensitive, accurate, low cost, and oneself qualitatively can quantitatively detect intended transgenic albumen again.But the method needs to carry out fluorescence labeling, complicated operation.And micro-cantilever carrys out the specific binding of detectable antigens and antibody based on the change of surface stress, without the need to mark, simple to operate, and sensitivity is higher.
Micro-cantilever biochemical sensing technology is in recent years along with a kind of emerging technology that scanning probe microscopy (SPM) and MEMS (micro electro mechanical system) (MEMS) develop rapidly.When there being biochemical reaction to occur in the single side surface of micro-cantilever, the change of its surface stress can cause micro-cantilever to produce flexural deformation, can measure deformation values (Nat.Biotech.2001,19,856 by optics or electricity reading method; Chem.Rev.2008,108,522).Compare with traditional ELISA method, micro-cantilever immune sensing technology based on antibody modification has the following advantages: measure without the need to mark, highly sensitive, the large scale that easily realizes real-time online, high flux, parallel array, potentially for substituting existing enzyme-linked immunosorbent assay method, tool may be detected to the analysis of genetically modified organism and is of great significance.Immunosensor at present based on micro-cantilever is mainly used for detecting PSA(Biosens.Bioelectron., 2005, 20, 2157), CRP(Biosens.Bioelectron., 2004, 20, 269), scFv (Proc.Natl.Acad.Sci., 2005, 102, 14587), IgG(Biosens.Bioelectron., 2005, 21, 597), myoglobin (Sens.Actuators, B2006, 117, 332) and GST (Nanotechnology, 2007, 18, 125503) etc., but up to the present, also without any patent and the bibliographical information method based on the detection transgene protein (as insecticidal crystal protein Cry1Ab) of micro-cantilever.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of detection insecticidal crystal protein CrylAb based on micro-cantilever of novel unmarked, high sensitivity, high specific.
For solving the problems of the technologies described above, this invention exploits a kind of method of detection insecticidal crystal protein Cry1Ab based on micro-cantilever, comprising following steps:
The micro-cantilever of the golden film of band is carried out pre-treatment, immerses in the alcohol solution of sulfhydrylization reagent, make sulfhydrylization reagent modify on the golden film of micro-cantilever, cleaning; Described cleaning comprises with ethanol purge, and nitrogen dries up;
Micro-cantilever is immersed in the mixed solution of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) and N-hydroxy-succinamide (NHS), make the activated carboxylic of sulfhydrylization reagent, cleaning; Described cleaning comprises by washed with de-ionized water, cleans with biological buffer solutions;
Immersed by micro-cantilever in the biological buffer solutions of insecticidal crystal protein Cry1Ab antibody, described biological buffer solutions comprises the PBS damping fluid of pH 7.4, leaves standstill, the amino of the carboxyl after activation and insecticidal crystal protein Cry1Ab antibody is combined.
The micro-cantilever of the above-mentioned Cry1Ab of being modified with antibody is used for the detection to insecticidal crystal protein Cry1Ab.
Further, described sulfhydrylization reagent is 11-Mercaptoundecanoic acid, 6-mercaptohexanoic acid or 3-mercaptopropionic acid, and concentration is 1 × 10
-4m ~ 1 × 10
-3m; Described concentration is preferably 1 × 10
-4m.
Further, the condition of the alcohol solution of described immersion sulfhydrylization reagent is: the time is 12h ~ 24h, and temperature is 15 DEG C ~ 37.5 DEG C; The described time is preferably 12h, and described temperature is preferably 15 DEG C
Further, described pre-treatment comprises by washed with de-ionized water, and nitrogen dries up.
Further, described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine hydrochloride concentration is 10mg/ml ~ 100mg/ml, is preferably 10mg/ml; The concentration of described N-hydroxy-succinamide is 10mg/ml ~ 100mg/ml, is preferably 10mg/ml; The volume ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1 ~ 5:1; Described volume ratio is preferably 1:1; The reaction time of described immersion is 0.5h ~ 4h; The described reaction time is preferably 4h.
Further, the concentration of described insecticidal crystal protein Cry1Ab antibody is 0.1mg/ml ~ 1mg/ml, is preferably 0.1mg/ml.
Further, described standing condition is: the time is 4h ~ 8h, is preferably 4h.
The present invention has following beneficial effect:
1, the present invention has very high specificity and sensitivity to the detection of insecticidal crystal protein Cry1Ab in genetically modified plants, lowest detectable limit can reach 0.4pg/ml, lower than the detectability (7.8ngml ~ 2.9pg/ml) of existing insecticidal crystal protein Cry1Ab enzyme-linked immunosorbent assay method.
2, method of the present invention is without the need to mark, simple to operate, highly sensitive, may be used for alternative existing enzyme-linked immunosorbent assay method, detect tool be of great significance the analysis of genetically modified organism.
Embodiment
Embodiment 1
1, micro-cantilever finishing insecticidal crystal protein Cry1Ab antibody
1) by the micro-cantilever (U.S. Mikromasch, long 350um, wide 35um, thick 1um, side is coated with the chromium of 20nm and the gold of 20nm) of the golden film of band at the ratio of amount of substance be the dense H of 7:3
2sO
4/ 30%H
2o
2mixed solution in soak 1min, use washed with de-ionized water afterwards three times, nitrogen dries up.Then micro-cantilever being immersed concentration is 1 × 10
-4in the ethanolic solution of the sulfhydrylization reagent 11-Mercaptoundecanoic acid of M, the time is 12h, and temperature is 15 DEG C, and take out and use ethanol purge three times, nitrogen dries up;
2) micro-cantilever of step 1) process is immersed in the mixed solution of EDCHCl and NHS, the concentration of EDCHCl and NHS is 10mg/ml, and the volume ratio of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1: 1; The reaction time of described immersion is 0.5h.Taking-up deionized water washes three times, then washes three times with the PBS buffer solution of pH 7.4;
3) antibody is connected
By through step 2) to immerse concentration be in the solution of insecticidal crystal protein Cry1Ab antibody of 0.1mg/ml for the micro-cantilever that processed, add PBS solution, the PBS buffer solution leaving standstill 4h, taking-up pH 7.4 under lucifuge condition washes three times, and the PBS buffer solution putting into pH 7.4 leaves standstill 1h.
2, micro-cantilever beam sensor sensitivity effect detection
(1) clean: washed with de-ionized water micro-cantilever beam sensor sample cell three times, then use ethanol purge three times, nitrogen dries up.
(2) sample solution: with the PBS buffer solution configuration Cry1Ab protein solution of pH 7.4; Sample solution concentration is respectively 0.4pg/ml, 4pg/ml, 40pg/ml, 0.4ng/ml, 4ng/ml
(3) sample determination: insert in the sample cell of micro-cantilever beam sensor by the micro-cantilever being modified with Cry1Ab antibody, mobile phase is the PBS buffer solution of pH 7.4, and flow velocity is 1ml/h.When adding above-mentioned the configured sample solution of 1ml after micro-cantilever beam sensor signal stabilization respectively, under lucifuge condition, utilizing micro-cantilever beam sensor to monitor, obtaining the bending displacement curve of micro-cantilever.
(4) system balancing: after reaction experiment terminates, takes out micro-cantilever, with PBS balance sample cell system.After often measuring a concentration, take out micro-cantilever, then carry out new operation by (1).Each micro-cantilever being modified with Cry1Ab antibody only detects a kind of Cry1Ab protein solution of concentration.
Three repetitions are established in experiment, results averaged, result shows that the semi-girder bending displacement detecting variable concentrations insecticidal crystal protein Cry1Ab is 6.0nm, 10.1nm, 16.0nm, 25.2nm and 35.5nm, illustrate that the bending displacement that this micro-cantilever detects insecticidal crystal protein Cry1Ab increases along with the increase of the concentration of Cry1Ab, most low energy detects 0.4pg/ml Cry1Ab.
3, micro-cantilever beam sensor specific effect detects
(1) clean: washed with de-ionized water micro-cantilever beam sensor sample cell three times, then use ethanol purge three times, nitrogen dries up;
(2) sample solution: with PBS buffer solution dilution goat anti-human igg and the Cowpea Trypsin Inhibitor (CpTI) of pH 7.4; Sample solution concentration is 1 μ g/ml;
(3) sample determination: insert in the sample cell of micro-cantilever beam sensor by the micro-cantilever being modified with Cry1Ab antibody, mobile phase is the PBS buffer solution of pH 7.4, and flow velocity is 1ml/h.When adding above-mentioned the configured sample solution of 1ml after micro-cantilever beam sensor signal stabilization respectively, under lucifuge condition, utilizing micro-cantilever beam sensor to monitor, obtaining the bending displacement curve of micro-cantilever.
(4) system balancing: after reaction experiment terminates, takes out micro-cantilever, with PBS balance sample cell system.After often measuring a concentration, take out micro-cantilever, then carry out new operation by (1).Each micro-cantilever being modified with Cry1Ab antibody only detects a kind of protein solution.
Experiment establishes three repetitions, results averaged, and result shows that the semi-girder bending displacement detecting IgG and CpTI is all less than 3nm.Illustrate that the detection of this semi-girder to insecticidal crystal protein Cry1Ab has specificity.
Embodiment 2
1, micro-cantilever finishing insecticidal crystal protein Cry1Ab antibody
1) by the micro-cantilever (U.S. Mikromasch, long 350um, wide 35um, thick 1um, side is coated with the chromium of 20nm and the gold of 20nm) of the golden film of band at the ratio of amount of substance be the dense H of 7:3
2sO
4/ 30%H
2o
2mixed solution in soak 1min, use washed with de-ionized water afterwards three times, nitrogen dries up.Then micro-cantilever being immersed concentration is 1 × 10
-4in the ethanolic solution of the sulfhydrylization reagent 6-mercaptohexanoic acid of M, the time is 12h, and temperature is 15 DEG C, and take out and use ethanol purge three times, nitrogen dries up;
2) micro-cantilever of step 1) process is immersed in the mixed solution of EDCHCl and NHS, the concentration of EDCHCl and NHS is the volume ratio of 10mg/ml, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1:1; The reaction time of described immersion is 0.5h.Taking-up deionized water washes three times, then washes three times with the PBS buffer solution of pH 7.4;
3) antibody is connected
By through step 2) to immerse concentration be in the solution of insecticidal crystal protein Cry1Ab antibody of 0.1mg/ml for the micro-cantilever that processed, add PBS solution, the PBS buffer solution leaving standstill 4h, taking-up pH 7.4 under lucifuge condition washes three times, and the PBS buffer solution putting into pH 7.4 leaves standstill 1h.
2, micro-cantilever beam sensor sensitivity effect detection
(1) clean: washed with de-ionized water micro-cantilever beam sensor sample cell three times, then use ethanol purge three times, nitrogen dries up.
(2) sample solution: with the PBS buffer solution configuration Cry1Ab protein solution of pH 7.4; Sample solution concentration is respectively 0.4pg/ml, 4pg/ml, 40pg/ml, 0.4ng/ml, 4ng/ml
(3) sample determination: insert in the sample cell of micro-cantilever beam sensor by the micro-cantilever being modified with Cry1Ab antibody, mobile phase is the PBS buffer solution of pH 7.4, and flow velocity is 1ml/h.When adding above-mentioned the configured sample solution of 1ml after micro-cantilever beam sensor signal stabilization respectively, utilizing micro-cantilever beam sensor to monitor, obtaining the bending displacement curve of micro-cantilever.
(4) system balancing: after reaction experiment terminates, takes out micro-cantilever, with PBS balance sample cell system.After often measuring a concentration, take out micro-cantilever, then carry out new operation by (1).Each micro-cantilever being modified with Cry1Ab antibody only detects a kind of Cry1Ab protein solution of concentration.
Three repetitions are established in experiment, results averaged, result shows that the semi-girder bending displacement detecting variable concentrations insecticidal crystal protein Cry1Ab is 3.5nm, 7.2nm, 11.0nm, 18.2nm and 30.1nm, illustrate that the bending displacement that this micro-cantilever detects insecticidal crystal protein Cry1Ab increases along with the increase of the concentration of Cry1Ab, most low energy detects 0.4pg/ml Cry1Ab.
3, micro-cantilever beam sensor specific effect detects
(1) clean: washed with de-ionized water micro-cantilever beam sensor sample cell three times, then use ethanol purge three times, nitrogen dries up;
(2) sample solution: with PBS buffer solution dilution goat anti-human igg and the Cowpea Trypsin Inhibitor (CpTI) of pH 7.4; Sample solution concentration is 1 μ g/ml;
(3) sample determination: insert in the sample cell of micro-cantilever beam sensor by the micro-cantilever being modified with Cry1Ab antibody, mobile phase is the PBS buffer solution of pH 7.4, and flow velocity is 1ml/h.When adding above-mentioned the configured sample solution of 1ml after micro-cantilever beam sensor signal stabilization respectively, utilizing micro-cantilever beam sensor to monitor, obtaining the bending displacement curve of micro-cantilever.
(4) system balancing: after reaction experiment terminates, takes out micro-cantilever, with PBS balance sample cell system.After often measuring a concentration, take out micro-cantilever, then carry out new operation by (1).Each micro-cantilever being modified with Cry1Ab antibody only detects a kind of protein solution.
Experiment establishes three repetitions, results averaged, and result shows that the semi-girder bending displacement detecting IgG and CpTI is all less than 3nm.Illustrate that the detection of this semi-girder to insecticidal crystal protein Cry1Ab has specificity.
Embodiment 3
1, micro-cantilever finishing insecticidal crystal protein Cry1Ab antibody
1) by the micro-cantilever (U.S. Mikromasch, long 350um, wide 35um, thick 1um, side is coated with the chromium of 20nm and the gold of 20nm) of the golden film of band at the ratio of amount of substance be the dense H of 7:3
2sO
4/ 30%H
2o
2mixed solution in soak 1min, use washed with de-ionized water afterwards three times, nitrogen dries up.Then micro-cantilever being immersed concentration is 1 × 10
-4in the ethanolic solution of the sulfhydrylization reagent 3-mercaptopropionic acid of M, the time is 12h, and temperature is 15 DEG C, and take out and use ethanol purge three times, nitrogen dries up;
2) micro-cantilever of step 1) process is immersed in the mixed solution of EDCHCl and NHS, the concentration of EDCHCl and NHS is the volume ratio of 10mg/ml, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1:1; The reaction time of described immersion is 0.5h.Taking-up deionized water washes three times, then washes three times with the PBS buffer solution of pH 7.4;
3) antibody is connected
By through step 2) to immerse concentration be in the solution of insecticidal crystal protein Cry1Ab antibody of 1mg/ml for the micro-cantilever that processed, add PBS solution, the PBS buffer solution leaving standstill 4h, taking-up pH 7.4 under lucifuge condition washes three times, and the PBS buffer solution putting into pH 7.4 leaves standstill 1h.
2, micro-cantilever beam sensor sensitivity effect detection
(1) clean: washed with de-ionized water micro-cantilever beam sensor sample cell three times, then use ethanol purge three times, nitrogen dries up.
(2) sample solution: with the PBS buffer solution configuration Cry1Ab protein solution of pH 7.4; Sample solution concentration is respectively 0.4pg/ml, 4pg/ml, 40pg/ml, 0.4ng/ml, 4ng/ml
(3) sample determination: insert in the sample cell of micro-cantilever beam sensor by the micro-cantilever being modified with Cry1Ab antibody, mobile phase is the PBS buffer solution of pH 7.4, and flow velocity is 1ml/h.When adding above-mentioned the configured sample solution of 1ml after micro-cantilever beam sensor signal stabilization respectively, utilizing micro-cantilever beam sensor to monitor, obtaining the bending displacement curve of micro-cantilever.
(4) system balancing: after reaction experiment terminates, takes out micro-cantilever, with PBS balance sample cell system.After often measuring a concentration, take out micro-cantilever, then carry out new operation by (1).Each micro-cantilever being modified with Cry1Ab antibody only detects a kind of Cry1Ab protein solution of concentration.
Three repetitions are established in experiment, results averaged, result shows that the semi-girder bending displacement detecting variable concentrations insecticidal crystal protein Cry1Ab is 0nm, 5.1nm, 10.0nm, 13.2nm and 24.3nm, illustrate that the bending displacement that this micro-cantilever detects insecticidal crystal protein Cry1Ab increases along with the increase of the concentration of Cry1Ab, most low energy detects 4pg/ml Cry1Ab.
3, micro-cantilever beam sensor specific effect detects
(1) clean: washed with de-ionized water micro-cantilever beam sensor sample cell three times, then use ethanol purge three times, nitrogen dries up;
(2) sample solution: with PBS buffer solution dilution goat anti-human igg and the cowpea trypsin inhibitor gene (CpTI) of pH 7.4; Sample solution concentration is 1 μ g/ml;
(3) sample determination: insert in the sample cell of micro-cantilever beam sensor by the micro-cantilever being modified with Cry1Ab antibody, mobile phase is the PBS buffer solution of pH 7.4, and flow velocity is 1ml/h.When adding above-mentioned the configured sample solution of 1ml after micro-cantilever beam sensor signal stabilization respectively, under lucifuge condition, utilizing micro-cantilever beam sensor to monitor, obtaining the bending displacement curve of micro-cantilever.
(4) system balancing: after reaction experiment terminates, takes out micro-cantilever, with PBS balance sample cell system.After often measuring a concentration, take out micro-cantilever, then carry out new operation by (1).Each micro-cantilever being modified with Cry1Ab antibody only detects a kind of protein solution.
Experiment establishes three repetitions, results averaged, and result shows that the semi-girder bending displacement detecting IgG and CpTI is all less than 3nm.Illustrate that the detection of this semi-girder to insecticidal crystal protein Cry1Ab has specificity.
The present invention can reach technique effect of the present invention in cited concentration, modification time, ratio and temperature range.
According to Chinese patent CN 102095855(Anal.Bioanal.Chem., 2010,396,2203-2211, J.Agric.Food.Chem., 2011,59,2184-2189) report, the detection of existing insecticidal crystal protein Cry1Ab enzyme-linked immunosorbent assay method is limited to 7.8ng/ml ~ 2.9pg/ml.Can be found out by above-described embodiment, the present invention has very high specificity and sensitivity to the detection of insecticidal crystal protein Cry1Ab in genetically modified plants, lowest detectable limit can reach 0.4pg/ml, lower than the detectability scope of existing insecticidal crystal protein Cry1Ab enzyme-linked immunosorbent assay method, there is high sensitivity.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.
Claims (7)
1., based on a method of the detection insecticidal crystal protein Cry1Ab of micro-cantilever, it is characterized in that, the method includes the steps of:
The micro-cantilever of the golden film of band is carried out pre-treatment, immerses in the alcohol solution of sulfhydrylization reagent, cleaning;
Micro-cantilever is immersed in the mixed solution of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide, cleaning;
Micro-cantilever is immersed in the biological buffer solutions of insecticidal crystal protein Cry1Ab antibody, leave standstill under lucifuge condition;
Under lucifuge condition, the micro-cantilever of the above-mentioned Cry1Ab of being modified with antibody is used for the detection to insecticidal crystal protein Cry1Ab;
Further, the concentration of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is 10mg/ml ~ 100mg/ml; The concentration of described N-hydroxy-succinamide is 10mg/ml ~ 100mg/ml; The volume ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1 ~ 5:1; The reaction time of described immersion is 0.5h ~ 4h.
2. method according to claim 1, is characterized in that, described sulfhydrylization reagent is 11-Mercaptoundecanoic acid, 6-mercaptohexanoic acid or 3-mercaptopropionic acid, and concentration is 1 × 10
-4m ~ 1 × 10
-3m.
3. method according to claim 1, is characterized in that, the condition in the alcohol solution of described immersion sulfhydrylization reagent is: the time is 12h ~ 24h, and temperature is 15 DEG C ~ 37.5 DEG C.
4. method according to claim 1, is characterized in that, described pre-treatment is: at dense H
2sO
4/ 30%H
2o
2mixed solution in soak 1min; Described dense H
2sO
4: 30%H
2o
2the ratio of amount of substance is 7:3.
5. method according to claim 1, is characterized in that, described pre-treatment also comprises by washed with de-ionized water, and nitrogen dries up.
6. method according to claim 1, is characterized in that, the concentration of described insecticidal crystal protein Cry1Ab antibody is 0.1mg/ml ~ 1mg/ml.
7. method according to claim 1, is characterized in that, the described standing time is 4h ~ 8h.
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